The long-term objective of this application is to understand the cellular mechanisms underlying the retrograde (minus-end) transport of mitochondria in cells. It is well established that kinesin heavy chain (KHC) directs anterograde (plus-end) movement of mitochondria, and that mitochondrial association of KHC requires the adaptor proteins Miro and Milton (Glater etal. 2006). While there have been great strides in defining the protein complex needed for anterograde transport of mitochondria, how retrograde movement of mitochondria is coordinated remains unclear. As with anterograde movement, retrograde movement of mitochondria is primarily microtubule dependent and presumably driven by the cytoplasmic motor dynein and the accessory protein complex dynactin. The focus of this proposal is to identify the cellular core machinery involved in regulating retrograde movement of mitochondria. More specifically, this proposal will investigate if Miro and Milton are required for dynein/dynactin association with mitochondria in cells. Understanding the regulation of mitochondrial motility is of significant importance, not only to the field of mitochondrial transport, but for neuronal disease. Several neurodegenerative diseases, such as Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SBMA), and Huntington's disease have been linked to defects in retrograde movement of organelles, including mitochondria (Strom et al. 2008;Hafezparast et al. 2003;Petrozzi et al. 2007). Preliminary studies from our laboratory indicate that anterograde and retrograde motors are simultaneously bound to mitochondria, and directionality is determined by a coordinated regulation of motors. Additionally, we have evidence that Miro, which contains two GTPase domains and two calcium-binding EF-hands, may serve as a calcium-sensitive linker for mitochondrial association of both kinesin and the retrograde motor complex. Using biochemical and live-imaging techniques, I intend to first investigate the in vivo localization of dynein/dynactin subunits on mitochondria. It will be interesting to determine whether dynein/dynactin associates with both anterograde and retrograde moving mitochondria, or whether dynein/dynactin association regulates the direction of movement. I will also examine whether Miro or Milton directly interacts with components of the retrograde motor complex by performing co-immunoprecipitation experiments from mammalian neuronal and cell cultures. In summary, understanding how Miro and Milton regulate transport of mitochondria provides a unique opportunity to investigate the regulation and bidirectional coordination of cellular motors and their cargoes. More importantly, gaining insight into how mitochondria are directed for transport into distal regions of axons will aide in understanding how defects in delivery of mitochondria lead to axonal degeneration and how the proper localization and delivery of mitochondria in cells contributes to normal cell functioning. PUBLIC HEALTH RELEVANCE: The proper localization and distribution of mitochondria is particularly vital in neurons, and several studies have linked the defective regulation and transport of mitochondria as significant contributors to neurodegenerative diseases, such as axonal Charcot-Marie-Tooth syndrome autosomal-dominant optic atrophy hereditary spastic paraplegia type 7, and amyotrophic lateral sclerosis (Zhao et al. 2001;Ferreirinha et al. 2004;Baloh et al. 2007;Vande Velde et al. 2008). Thus, there is a need for further detailed understanding of how cells regulate the delicate distribution of mitochondria. Understanding the mechanisms underlying motility of axonal mitochondria will yield important clues on the regulation of general organelle transport, as well as provide a deeper understanding of how defects in the physiological regulation of mitochondrial transport may lead to oxidative damage, metabolic insufficiency, and neurodegenerative disease.